Biol. Chem. 2015; aop Review Jan Riemer * , Markus Schwarzl ä nder * , Marcus Conrad * and Johannes M. Herrmann * Thiol switches in mitochondria: operation and physiological relevance Abstract : Mitochondria are a major source of reactive Introduction – mitochondria oxygen species (ROS) in the cell, particularly of super- oxide and hydrogen peroxide. A number of dedicated contain two distinct redox networks enzymes regulate the conversion and consumption of superoxide and hydrogen peroxide in the intermem- Mitochondria are essential organelles of eukaryotic cells. brane space and the matrix of mitochondria. Neverthe- They produce not only the bulk of cellular energy in the less, hydrogen peroxide can also interact with many other form of ATP, but they also generate numerous important mitochondrial enzymes, particularly those with reactive metabolites and cofactors, and they serve as critical sign- cysteine residues, modulating their reactivity in accord- aling stations that, for example, integrate cellular signals ance with changes in redox conditions. In this review we to initiate apoptosis. In turn, mitochondria also commu- will describe the general redox systems in mitochondria nicate their metabolic and fitness state to the remainder of animals, fungi and plants and discuss potential target of the cell to trigger cellular adaptation processes. proteins that were proposed to contain regulatory thiol Mitochondria contain two distinct aqueous sub- switches. compartments, the intermembrane space (IMS) and the matrix. Both subcompartments differ strongly with Keywords: glutathione; hydrogen peroxide; mitochon- respect to their biological activity, their protein composi- dria; NADPH; reactive oxygen species; redox regulation; tion ( Herrmann and Riemer, 2010 ) as well as their redox ROS; signaling; thiol switch. properties. Firstly, whereas the IMS is connected to the cytosol via porins, which allow the free diffusion of mol- ecules of up to 5 kDa (including GSH/GSSG, NADPH/ DOI 10.1515/hsz-2014-0293 + Received December 2 , 2014 ; accepted January 19 , 2015 NADP or hydrogen peroxide), the matrix is strictly sepa- rated from the IMS as the transport across the inner mem- brane is tightly controlled by substrate-specific carriers. Secondly, most cysteine residues of matrix proteins are believed to be reduced in the matrix, while many IMS pro- teins contain structural disulfide bonds which are intro- duced by the mitochondrial disulfide relay (also called MIA pathway) ( Chacinska et al., 2004 ; Naoe et al., 2004 ; Allen et al., 2005 ; Mesecke et al., 2005 ; Bihlmaier et al., 2007 ; Banci et al., 2009 ; Kawano et al., 2009 ; Milenko- *Corresponding authors: Jan Riemer, Cellular Biochemistry, vic et al., 2009 ; Bien et al., 2010 ; von der Malsburg et al., University of Kaiserslautern, Erwin-Schr ö dinger-Str. 13, D-67663 2011 ; Fischer et al., 2013 ; Koch and Schmid, 2014 ). Kaiserslautern, Germany, e-mail: [email protected] ; The IMS may even be considered as two separate Markus Schwarzl ä nder, INRES – Chemical Signalling, University of Bonn, Friedrich-Ebert-Allee 144, D-53113 Bonn, Germany, subcompartments because the peripheral IMS, which is e-mail: [email protected]; Marcus Conrad, adjacent to the outer membrane, is separated from the Institute of Developmental Genetics, Helmholtz Center Munich, cristae space by cristae junctions ( Frey and Mannella, German Research Center for Environmental Health, Ingolst ä dter 2000 ; Scorrano et al., 2002 ). Recently, a protein complex Landstra ß e 1, D-85764 Neuherberg, Germany, termed MICOS was identified and found to be located e-mail: [email protected]; and Johannes M. Herrmann, Cell Biology, University of Kaiserslautern, Erwin- in the inner membrane and is critical for the separation Schr ö dinger-Str. 13, D-67633 Kaiserslautern, Germany, of these two subcompartments as well as for mitochon- e-mail: [email protected] drial functionality ( Harner et al., 2011 ; Hoppins et al., Bereitgestellt von | Helmholtz Zentrum Muenchen - Deutsches Forschungszentrum fuer Gesundheit und Umwelt Angemeldet Heruntergeladen am | 27.03.15 10:29 2 J. Riemer et al.: Thiol switches in mitochondria 2011 ; von der Malsburg et al., 2011 ; K ö rner et al., 2012 ). metabolism that, over time, cause deleterious oxidative Cristae junctions restrict (and perhaps even control) the stress in eukaryotic cells. However, this traditional concept diffusion of molecules into and out of the cristae. Hence, has been challenged by many recent studies which dem- the small molecule environment of the cristae space onstrated that ROS (in particular hydrogen peroxide) also – specifically with regards to its pH, glutathione redox serve as critical signaling molecules ( Chandel et al., 2000 ; potential and levels of reactive oxygen species (ROS) – Albrecht et al., 2011 ; Zarse et al., 2012 ; Mouchiroud et al., likely differs from that of the peripheral IMS and is more 2013 ; Gladyshev, 2014 ; Sies, 2014 ; Yee et al., 2014 ). strongly influenced by the respiratory chain, which is ROS encompass a variety of different molecules • - mainly localized in cristae membranes. There is good evi- including the superoxide radical (O2 ), hydrogen per- • dence that proteins of the IMS and the inner membrane oxide (H2 O2 ) and the hydroxyl radical ( OH) that are are partitioned between the cristae and the peripheral derived from the partial reduction of molecular oxygen inner membrane but the mechanisms by which segrega- (O2 ). ROS can also result from the ‘ detoxification ’ of other • - tion takes place are unknown ( Vogel et al., 2006 ; Wurm ROS, as is the case during the dismutation of O2 to H2 O2 and Jakobs, 2006 ; Stoldt et al., 2012 ; Daum et al., 2013 ). (and oxygen). H2 O2 is a particularly suitable candidate The mitochondrial matrix is separated from the IMS by to act as a signaling molecule, because of its relatively the inner membrane, which only allows a tightly regulated long half-life ( Sies, 1993 ), relatively high concentra- exchange of small molecules. Thus, the small molecule tion (nanomolar to low micromolar), and reasonable environment of the matrix is mainly controlled indepen- membrane permeability. In mitochondria channels in dently from the remainder of the cell. This is nicely illus- the outer membrane (porins) and the inner membrane, trated for example by the regulation of GSH/GSSG ratio in mitochondrial aquaporin-8 might considerably acceler- the matrix ( Hu et al., 2008 ; Kojer et al., 2012 ). The matrix ate the exchange of hydrogen peroxide ( Calamita et al., GSH/GSSG ratio is similar to its cytosolic counterpart, but 2005 ; Marchissio et al., 2012 ). Perhaps most importantly, is subject to completely different regulatory machineries H2 O2 can selectively oxidize specific cysteine residues in including mitochondria-specific ROS-generating path- target proteins, making it an ideal candidate as a sign- ways (see below) and a partly mitochondria-specific set aling molecule ( Winterbourn and Hampton, 2008 ). H2 O2 of reducing enzymes, including factors for the reductive can oxidize thiols to sulfenic, sulfinic and sulfonic acid, regeneration of NADPH and GSH. The differences between of which the first two can be enzymatically converted the mitochondrial compartments lead to differences in back to thiols. Sulfenic acid can react with another thiol the generation, handling and perception of oxidizing and to a disulfide. In contrast, • OH is highly reactive (reacting reductive influences. with diffusion-limited rate constants) and will indiscrim- In the following we will illustrate this notion by inately oxidize biomolecules, rendering it unsuitable as describing and discussing the mechanisms that drive a signaling molecule. the reversible operation of molecular cysteine switches. Mitochondrial pathways are major sources of cel- Molecular switch operating is driven by the balance of lular H2 O2 ( Figure 2 ). Different complexes of the respira- • - opposing influences ( Figure 1 ). Oxidizing influences tory chain release O2 towards both the matrix and the mainly stem from the production of ROS at different sites IMS ( Murphy, 2009 ; Bleier et al., 2014 ; Dr ö se et al., 2014 ). • - in mitochondria; reducing influences ultimately originate O2 is rapidly converted to H2 O2 by superoxide dismutases from NADPH, which is regenerated by different metabolic (copper-zinc SOD in the IMS and cytosol, manganese SOD ∼ • - pathways. Both thiol switch drivers rely on enzymatic in the matrix). The 10 000 fold increase in the rate of O 2 machineries for the modulation and the relay of signals. dismutation by SODs not only accelerates the production of H2 O2 , but also competes with the production of perox- - • - • initrite (ONOO ) by the reaction of O2 with NO . While dedicated NO synthases have been reported in mamma- Redox influences and redox lian mitochondria ( Giulivi et al., 1998 ; Nisoli et al., 2003 ), enzymes in mitochondria reports of a dedicated NO synthase in plant mitochondria have turned out to be incorrect ( Gas et al., 2009 ); instead a likely source of mitochondrial NO is the mitochondrial Oxidizing influences in mitochondria respiratory chain by nitrite reduction ( Gupta et al., 2011 ). - Both H2 O2 and ONOO represent major electron sinks for Endogenous ROS have for a long time been considered as the matrix thiol machinery and are likely to be main drivers the unwanted but unavoidable downside of mitochondrial behind thiol oxidation, both directly and indirectly.